This was the second year of the project. During this year: 1. We studied the genomic response of five primary human immune cells (B lymphocytes, CD4+ and CD8+ T lymphocytes, monocytes, and neutrophils) and six primary human non-immune cells (fibroblasts, endothelial cells, myoblasts, osteoblasts, pre-adipocytes, and mature adipocytes) exposed in vitro to the glucocorticoid methylprednisolone and sampled serially after the exposure. We identified the genes and pathways that respond to the glucocorticoid stimulus in each cell type. A subset of genes with known immune function responds to the glucocorticoid stimulus in each immune cell type, but not in the non-immune cell types. Furthermore, we have identified genes that respond to the glucocorticoid stimulus in immune cells, but are not expressed at all in the non-immune cells studied. These genes, and the molecular pathways in which they are involved, represent an initial set of potential targets for future therapeutic interventions aimed at reproducing the rapid and potent anti-inflammatory and immunosuppressive actions of glucocorticoids, without the extensive side effects that glucocorticoids have on non-immune tissues. 2. We conceived, wrote, and passed through the NIH scientific and regulatory review process a clinical study aimed at understanding the genomic response to glucocorticoids in human immune and non-immune cells in vivo. In this study, we will recruit a cohort of healthy volunteers at the NIH Clinical Centers Day Hospital. Volunteers will receive a single intravenous dose of the glucocorticoid methylprednisolone. We will study the in vivo genomic response to the glucocorticoid in six immune cell types (B lymphocytes, CD4+ and CD8+ T lymphocytes, monocytes, neutrophils, and NK cells) and one primarily non-immune tissue (skin), with serial sampling. We expect the results of this initial human study to complement the in vitro work of the past year, advancing towards our goal of generating a detailed understanding of the genes and pathways that are responsible for the anti-inflammatory and immunosuppressive effects of glucocorticoids, and how these differ from the effects of this group of drugs on non-immune cells. The study has been approved by the Scientific Review Committee, the Regulatory Review Group, and the Institutional Review Board, and is expected to start recruitment in the second half of 2016. 3. In collaboration with Dr. Amy Klions laboratory, we began a study of the effects of glucocorticoids on human eosinophils. Glucocorticoids are extensively used to treat human diseases that involve increased numbers or activity of eosinophils, but similar to other indications their exact mechanism of action in patients with eosinophilia is not known. We recruited an initial cohort of five patients with benign hypereosinophilia, a condition in which circulating levels of eosinophils are elevated. We administered a single dose of the glucocorticoid prednisone and described the kinetics of the observed drop in circulating eosinophils, finding a consistent pattern of decline between two and three hours after administration. It remains unclear whether this drop occurs in response to molecular changes within the eosinophils or in other cell types (endothelial or reticuloendothelial cells, for example). To understand the molecular changes that occur within eosinophils in the two hours preceding the observed decline, we have performed sequencing of total RNA in eosinophils from these five subjects, after serial sampling. Analysis of this data is ongoing. To study the distribution of circulating eosinophils and the possibility of effects in cells other than eosinophils, we have begun to work with Dr. Peter Choyke (NCI) on live cell tracking experiments, and with Dr. Cynthia Dunbar (NHLBI) on the possibility of using animal models to advance this work.